The present invention relates to a composition for forming an electrode, an electrochemical sensor comprising the same, and a method for determining an analyte using the electrochemical sensor.
Measuring systems for biochemical analysis are important components of clinically relevant analytical methods. This primarily concerns the measurement of analytes which can be directly or indirectly determined with the aid of enzymes. Determination of the concentration of clinically useful parameters is generally carried out using in vitro analytical systems. However, when determining analytes which show a significant change in their concentration during the course of the day an in vitro analysis is inappropriate due to limited temporal resolution and the difficulties encountered with sampling.
In this case, biosensors, i.e., measuring systems equipped with biological components, which allow a repeated measurement of the analyte either continuously or discontinuously and which can be used ex vivo as well as in vivo have proven to be particularly suitable for the measurement of analytes. Ex vivo biosensors are typically used in flow-through cells whereas in vivo biosensors can, for example, be implanted into subcutaneous fatty tissue. In this connection one distinguishes between transcutaneous implants which are only introduced into the tissue for a short period and are in direct contact with a measuring device located on the skin, and full implants which are inserted surgically into the tissue together with a measuring device.
Electrochemical biosensors which comprise an enzyme as a biological component contain the enzyme in or on the working electrode in which case for example the analyte can serve as a substrate for the enzyme and can be physicochemically altered (e.g. oxidized) with the aid of this enzyme. The electrical measuring signal generated by the flow of electrons released during conversion of the analyte on the working electrode correlates with the concentration of the measured analyte such that the electrical measuring signal can be used to determine the presence and/or the amount of the analyte in the sample.
In practice, a working electrode must fulfill a number of requirements in order to be suitable in electrochemical sensors:
The working electrode should have a low contact resistance and hence should be highly conductive.
The working electrode should not comprise any components which are electrochemically converted in the selected polarisation voltage. This can be accomplished by a suitable choice of binders and fillers.
The electrochemically active surface area of the working electrode has to be kept constant over its entire period of operation. For this purpose, a reduction of the surface area due to the adsorption of components of the surrounding fluid has to be avoided. This is generally affected by applying one or several polymer coatings, which are highly biocompatible.
The electrochemical reaction of the conversion product of the enzymatic reaction should be effected at a low overpotential in order to minimize the decomposition voltage and hence enable a specific conversion of the parameter. For this purpose, a fast transfer of electrons from the prosthetic group of the enzyme to the diverting electrode is to be provided.
The working electrode should comprise a sufficient amount of analyte-specific enzyme having sufficient and constant activity in order to guarantee that the enzymatic reaction superposed to the electrochemical conversion is not limited by the available enzymatic activity, but by the available amount of analyte. In other words, the sensitivity has to be maintained throughout the entire period of operation. A diffusion of the enzyme from the working electrode into the surrounding tissue has to be avoided, also for the reason of a possible toxicity of the enzyme. Finally, it has to be provided for that the enzymatic activity does not fall below a predetermined limit during storage.
A plurality of electrode compositions is known for minimizing overpotentials. As regards H2O2, the oxidation potential can be reduced, for example, by 450 mV by using rhodium- and glucose oxidase-coated carbon fibres as compared to carbon fibres coated with glucose oxidase alone (Wang et al., Analytical Chemistry (1992), 64, 456-459). A method which is simpler to realize is described in EP 0 603 154 A2, which document provides an electrode composite produced by thoroughly mixing oxides and/or hydroxides of elements of the 4th period of the periodic table with graphite and a binder, leading to a reduction in overvoltage of the anodic H2O2 oxidation by >200 mV.
In addition to electrically nonconductive and/or semiconductive metal oxides generally introduced into electrode composites, electrically conductive electrocatalysts such as carbon nanotubes are known, which, due to their small size, may be arranged in proximity to the prosthetic centre of the enzyme, have a high electric conductivity and enable an efficient transfer of electrons (Wang et al., Analyst (2003), 128, 1382-1385; Wang et al., Analyst (2004), 129, 1-2; Wang et al., Analytica Chimica Acta (2005), 539, 209-213; Shobha Jeykumari et al., Biosensors and Bioelectronics (2008), 23, 1404-1411). Due to their high surface area, small amounts of nanotubes are sufficient to obtain a reduction of the decomposition potential (US 2006/0021881 A1).
Different measures are known in the art in order to provide for a constant enzyme activity. One possibility to avoid enzyme diffusion from the surface of the working electrode into the environment is to provide the working electrode with a suitable coating, e.g. a cover membrane. However, the use of such coatings in electrochemical sensors is associated with certain problems such as the necessity to deposit pinhole-free membranes. Secondly, the cover membrane must be deposited with a highly reproducible layer thickness for mass transfer limited systems. This requirement represents an extensive restriction of possible coatings since the realization of very thin layers exhibiting a sufficiently high barrier to mass transfer is difficult to realize.
Moreover, electrochemical sensors which are used to determine different analytes must usually also contain different cover membranes in order to provide different mass transfer rates of the substrate and co-substrate to the electrode. At the same time it must be ensured that the cover membranes are highly biocompatible for in vivo applications. Since even the smallest defects in the membrane are sufficient to result in a bleeding of the enzyme from the electrode into the environment, an enormous amount of quality control is necessary especially in the case of in vivo biosensors, resulting in considerable technical requirements and increased production costs.
Alternatively, the extent of enzyme bleeding can be reduced by immobilizing the enzyme in the electrode matrix of the working electrode which has led to an intensive search for suitable immobilization methods for enzymes in electrochemical biosensors. In practice, the enzyme may either be coupled to one or more paste components in a chemically covalent manner or be inserted physically into a composite so that the enzyme is adsorptively bonded to one or several paste components and/or is incorporated therein.
As regards adsorptive immobilization, Rege et al. (Nano Letters (2003), 3, 829-832) disclose an electrode composite comprising single-wall carbon nanotubes (SWCNT) and/or graphite as conductive fillers and PMMA as binder, wherein chymotrypsin is physically retained. In this context, it was found out that SWCNT-containing composites show less enzyme bleeding than graphite-containing ones, which seems to be due to a higher surface energy of the SWCNTs as compared to graphite.
Tang et al. (Analytical Biochemistry (2004), 331, 89-97) showed that by using a CNT-electrode, onto which Pt particles were electrochemically deposited and which was adsorptively modified with glucose oxidase, the long-term stability of glucose oxidase can be significantly increased as compared to a conventional graphite electrode.
Tsai et al. (Langmuir (2005), 21, 3653-3658) disclose a stable glucose sensor which comprises a carbon electrode coated with a composite containing multi-wall carbon nanotubes (MWCNTs), Nafion and glucose oxidase. The immobilizing effect of the matrix is referred to the electrostatic interaction of negatively charged MWCNTs and Nafion on the one hand, and positively charged glucose oxidase on the other hand.
Guan et al. (Biosensors and Bioelectronics (2005), 21, 508-512) realize a glucose sensor by dispensing a MWCNT-containing suspension and a GOD- and a ferricyanide-containing solution onto a screenprinted carbon electrode. An increased linearity of the response curve was observed and attributed to an increased electron transfer rate of the MWCNTs as compared to that of a conventional carbon electrode.
Kurusu et al. (Analytical Letters (2006), 39, 903-911), finally, disclose that the use of an electrode comprising a mixture of MWCNT, GOD and mineral oil leads to a significant reduction of the oxidative decomposition voltage of H2O2.
However, adsorptive immobilization procedures suffer from a number of problems. A major disadvantage of physical immobilization is the dependency of the binding constant on the composition of the medium surrounding the electrode, requiring a barrier membrane to prevent enzyme leakage.
In particular, however, physical coupling makes heavy demands on the reproducibility of the effective surface of the working electrode and, thus, on the production thereof. As described above, adsorptive immobilization either requires an application of enzyme-containing solutions and/or suspensions onto the surface of a prefabricated working electrode or an introduction of enzyme-containing solutions and/or suspensions into an electrode composite. The dispensing application of enzyme-containing solutions and/or suspensions onto the surface of a working electrode, however, has the disadvantage that an addition of small volumes of enzyme solution, for example a volume in the range of nanoliters, makes high demands on the precision of an automated dosing apparatus. Furthermore, the distribution of enzyme on the surface of the working electrode and transfer of enzyme into the pores of the working electrode depends on the topography and the energy of the electrode's surface, which is difficult to reproduce.
In view of the above, the admixture of enzymes into an electrode composite is to be preferred. However, a loss of the effective enzyme activity caused by shearing, the influence of solvents and thermal impact cannot be avoided due to the requirement of a homogeneous distribution of enzymes in mostly hydrophobic composites. Moreover, restrictions with respect to overall paste conductivity and adhesion onto the substrate have to be taken into account since specific rheological characteristics are required for the deposition of the electrode paste, while a constant consistency of the paste after homogeneous distribution of a dry enzyme in the composite has to be provided.
As an alternative, the enzyme may be introduced into the composite in an aqueous solution or in a suspension in order to minimize denaturation. This, however, brings about the necessity of a subsequent removal of solvent or suspension agent so that the composite cannot be supposed to have constant rheological characteristics over the production period.
Hence, in view of the disadvantages of an adsorptive immobilization there is thus a concrete need to immobilize enzymes in electrochemical biosensors by covalent bonds to at least one component of the electrode matrix.
EP 0 247 850 A1 discloses biosensors for the amperometric detection of an analyte. These sensors contain electrodes with immobilized enzymes which are immobilized or adsorbed onto the surface of an electrically conducting support where the support consists of a platinized porous layer of resin-bound carbon or graphite particles or contains such a layer. For this purpose, electrodes made of platinized graphite and a polymeric binding agent are firstly prepared and these are subsequently brought into contact with the enzyme. In this case, the enzyme is immobilized either by adsorption to the electrode surface or by coupling it to the polymeric binding agent using suitable reagents.
Amperometric biosensors with electrodes comprising an enzyme immobilized or adsorbed onto or into an electrically conducting, porous electrode material are also described in EP 0 603 154 A2. In order to produce the enzyme electrodes, an oxide or oxide hydrate of a transition metal of the fourth period, such as for example manganese dioxide, acting as a catalyst is worked into a paste together with graphite and a non-conducting polymeric binding agent, and the porous electrode material obtained after drying the paste is brought into contact with the enzyme in a second step. The enzyme can be immobilized on or in the porous electrode material by using a cross-linking reagent such as glutardialdehyde.
A major disadvantage of the electrochemical biosensors described in EP 0 247 850 A1 and EP 0 603 154 A2 is that the enzyme is first immobilized on the electrode that has been prefabricated without enzyme. As a consequence, there is the problem that the enzyme cannot be coupled to the electrode components in a controlled manner. Thus, when glutardialdehyde is used as a cross-linking reagent, the enzyme not only binds in an uncontrolled manner to any reactive components of the electrode material, but it is also inter-crosslinked. Furthermore, this procedure contaminates the electrode with the reagents that are used and, hence, the electrode has to again be thoroughly cleaned especially before use in an in vivo biosensor which increases the production complexity and thus the costs.
Cho et al. (Biotechnology and Bioengineering (1977), 19, 769-775) describe the immobilization of enzymes on activated carbons by covalent coupling. More particularly, the immobilization of glucose oxidase to petroleum-based activated carbons by (a) adsorption of the enzyme followed by cross-linking with glutaraldehyde or (b) activation of the carbon surface with a diimide and subsequent reaction with the enzyme is described. By this means, a considerably slower deactivation of the enzyme in the presence of H2O2 could be shown as compared to the soluble enzyme.
Li et al. (Analytical and Bioanalytical Chemistry (2005), 383, 918-922) disclose a glucose biosensor comprising a modified glassy carbon electrode as the working electrode. The modified glassy carbon electrode is prepared by coating a commercial electrode's surface with a dispersion of functionalized multi-wall carbon nanotubes (MWCNTs) having oxidized glucose oxidase covalently attached thereto in PBS buffer solution which contains Nafion® and ferrocene monocarboxylic acid. The catalytic effect of the functionalized nanotubes for glucose oxidation is particularly emphasized.
US 2007/0029195 A1 discloses immobilization of biomolecules such as proteins by covalent coupling to a conductive polymeric matrix reinforced by nanoparticles to improve the mechanical stability, electrical conductivity and immobilization of biomolecules. The matrix is a nanocomposite comprising gold nanoparticles coated with a polypyrrole formed from pyrrole and pyrrole propylic acid, wherein the latter compound provides for the covalent attachment of the biomolecules.
US 2008/0014471 A1 discloses electrochemical sensors comprising electrodes employing cross-linked enzyme clusters immobilized on carbon nanotubes (CNTs). In detail, the immobilization involves functionalization of the nanotubes' surface, subsequent covalent attachment of an enzyme such as glucose oxidase by means of a linking reagent to yield a CNT-enzyme conjugate, precipitation of free enzymes with a precipitation agent, and final treatment with a cross-linking reagent to form cross-linked enzyme clusters covalently attached to the CNTs via the CNT-enzyme conjugate.
US 2008/044911 A1 discloses a glucose sensor comprising nanowires having glucose oxidase covalently attached to their surface, which functionalized nanowires are prepared by contacting conventional nanowires with a linking reagent, e.g. a silane, and the enzyme. Conversion of glucose in a sample to be examined by the glucose oxidase immobilized on the nanowires' surface results in a change in pH of the test solution, wherein this change in pH produces a signal in the nanowires which can be detected by suitable means.
It is known in the art that covalent coupling of an enzyme to a support (e.g. MANAE-agarose, activated glyoxyl agarose and glutaraldehyde agarose) leads to a stabilization of the enzyme against thermal decomposition (Betancor et al., Journal of Biotechnology (2006), 121, 284-289). However, in addition to thermal decomposition, biosensors employing such immobilized enzymes also face the problem of enzyme decomposition caused by organic solvents during storage of the biosensor or caused by oxidative agents such as H2O2 during the period of operation.
In practice, biosensors must fulfill a plurality of requirements in order to allow exact measurements for immediate or time-delayed therapeutic measures. In particular, it is of uppermost importance that the analyte of interest can be determined with both a high specificity and sensitivity in order to enable determination of low amounts of the clinically relevant parameter. Consequently, the significant loss in enzymatic activity generally observed in commercially available biosensors when storing the same for more than one month is not acceptable for diagnostical and/or clinical purposes.